Project Summary/Abstract There is a critical need for improved in vitro disease modeling to rapidly advance therapeutic drug candidates to preclinical evaluation or to prioritize potential environmental toxicants. Recently, there have been significant advances made in in vitro disease models, including human mini-tissues derived from pluripotent stem cells (PSCs) and progenitor cells (a.k.a., organoid), bioprinted human tissue constructs with cells obtained from patients (a.k.a., 3D bioprinting), and multi-layered cells in microfluidic chips (a.k.a., organ-on-chip). These new and innovative technologies, however, still lack enough throughput and user friendliness to enable rapid identification of high-quality therapeutic candidates, particularly when a disease involves multiple organ interactions. To address these challenges, we propose to leverage our unique ?miniature three-dimensional (3D) bioprinting? technology and associated pillar/perfusion plate platforms, including a 384-pillar plate with sidewalls and slits (384PillarPlate) and a clear-bottom, 384-deep well plate (384DeepWellPlate) developed for static organoid culture as well as a 36-pillar plate with sidewalls and slits (36PillarPlate) and a 36- perfusion well plate with reservoirs and microchannels (36PerfusionPlate) for perfusion-based organoid culture. Our proposed pillar/perfusion plate platforms combining ?3D bioprinting? with ?microfluidic-like? features offer several distinctive advantages over more conventional 3D cell culture models and microfluidic models. In particular, the pillar/perfusion plates are compatible with standard 384-well plates and existing high-throughput screening (HTS) equipment (e.g., automated fluorescence microscopes and microtiter well plate readers) already familiar to users, which will significantly lower barriers to entry for commercialization. In the proposed research, human brain organoids (HBOs) derived from induced pluripotent stem cells (iPSCs) are selected as a model system to develop a predictive assessment tool for developmental neurotoxicity (DNT) by compounds including opioid drugs and alcohol. Our core hypotheses are: (i) bioprinted HBOs on the pillar/perfusion plates can maintain key tissue biomarkers by controlling and mimicking in vivo microenvironments and enable high- throughput, high-content cell function analysis; (ii) HBOs on the pillar/perfusion plates can model the influence of drugs and environmental toxicants to neurodevelopmental disorders. The specific aims of the proposed research are to: (1) improve reproducibility of organoid culture via miniature 3D bioprinting technology; (2) establish in situ whole organoid imaging on a pillar plate for high-throughput, predictive compound screening; (3) establish cryopreservation of organoids on the pillar plate. We envision that bioprinted human organoids on the pillar/perfusion plate platforms can be used as promising disease models for screening therapeutic drugs while minimizing the use of animals in drug discovery processes.